MX2011000401A - Silicon dioxide nanoparticles and their use for vaccination. - Google Patents

Silicon dioxide nanoparticles and their use for vaccination.

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Publication number
MX2011000401A
MX2011000401A MX2011000401A MX2011000401A MX2011000401A MX 2011000401 A MX2011000401 A MX 2011000401A MX 2011000401 A MX2011000401 A MX 2011000401A MX 2011000401 A MX2011000401 A MX 2011000401A MX 2011000401 A MX2011000401 A MX 2011000401A
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Mexico
Prior art keywords
nanoparticles
antigens
cells
antigen
targeting
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Application number
MX2011000401A
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Spanish (es)
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MX341881B (en
Inventor
Markus Weigandt
Andrea Hanefeld
Armin Kuebelbeck
Gregor Larbig
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Merck Patent Gmbh
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Application filed by Merck Patent Gmbh filed Critical Merck Patent Gmbh
Publication of MX2011000401A publication Critical patent/MX2011000401A/en
Publication of MX341881B publication Critical patent/MX341881B/en

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Abstract

The invention relates to ultra-small, monodisperse nanoparticles of silicon dioxide with at least one antigen attached to their surface. The nanoparticles can be used for the immunoprophylaxis or immunotherapy of cancer. The invention also provides a method of targeting antigens at antigen-presenting cells and of activating the immune system, the efficiency of targeting and/or immune activation being adjusted via the particle characteristics. The invention also relates to methods of active and passive immunization of a mammal.

Description

NANOPARTICLES OF SILICON DIOXIDE AND THE USE OF THEM FOR VACCINATION Description of the invention The invention relates to monodisperse, ultra-small nanoparticles comprising silicon dioxide on the surface of which at least one antigen binds. Nanoparticles can be used for immunoprophylaxis or cancer immunotherapy. The invention also relates to a method for the targeting of antigens in cells that present antigens and for activation of the immune system, where the efficiency of targeting and / or immunoactivation is adjusted via the characteristics of the particles . The invention also relates to a method for active and passive immunization of a mammal.
The health of a human animal or organism depends, inter alia, on the degree to which the organism is able to protect itself against pathogens from its environment or the degree to which the organism is able to recognize and eliminate modified endogenous material. The immune system of the human or animal body, which carries out these functions, can be divided into two functional areas: the innate and the immune system. the acquired immune system. Innate immunity is the first line of defense against infections and most of REF: 215888 potential pathogens become harmless before they are. capable of causing, for example, a detectable infection. The acquired immune system reacts to the surface structures of the entering organism or the modified endogenous material, which are known as antigens.
There are two types of immune responses acquired: the humoral immune response and the cell-mediated immune response. In the humoral immune response, the antibodies present in body fluids bind to antigens and initiate the deactivation thereof. In. the immune response mediated by cells, the T cells, which are capable of destroying other cells, become active. If, for example, the proteins associated with a disease are present in a cell, they are fragmented proteolytically within the cell to provide peptides. The specific cellular proteins are then bound to the resulting fragments of the protein or antigen and transport the latter to the cell surface, where they are presented to the molecular defense mechanisms, in particular the T cells of the body.
The molecules which carry the peptides to the cell surface and present them are known as proteins of the major histocompatibility complex (MHC, for its acronym in English). The importance of MHC proteins is also that they make it possible for T cells to distinguish endogenous antigens from exogenous antigens. Knowledge of the sequence of an exogenous peptide of this type makes it possible for the immune system to maneuver against diseased cells, for example using peptide vaccines.
The technologies for the presentation of proteogenic or peptide antigens in the area of vaccines must perform two fundamental tasks: the efficient transport of the antigen to the dendritic cells and the subsequent activation thereof in order to produce an acquired immune response. Current vaccine development focuses on molecular strategies which target peripheral dendritic cells, such as, for example, in the skin or muscles, as a target. The antigens are targeted to their dendritic destination by, in particular, antibodies which are specific for the cell surface receptors of the dendritic cells and are either fused to antigens or bound to surfaces of the particles. However, these demanding designs for cell-specific targeting are not necessary, as was shown by, inter alia, Fifis et al. (2004) J Immunol. 173 (5), 3148, who caused an immune response by transporting silver-conjugated polystyrene beads to the dendritic cells.
Furthermore, it is known in immunology to use adjuvants in order to increase the immune response not specifically to a given substance. In this way, while the antigen causes the specific immune response, the adjuvant is essentially responsible for the potency of this response. In order to cause an acquired immune response, the use of adjuvants is vital for the induction of dendritic cell maturation. Dendritic cells mature at this point as a result of molecular hazard signals, which act via signaling pathways of congenital immunity, such as, for example, Toll-like receptors (TLRs) or the inflammatory cytokine receptors. WO 2004/108072 A2, for example, describes a conjugate in which compounds which modify the immune response, such as, for example, TLR agonists, bind to a metal particle support, which further comprises at least one active compound. However, the compounds which modify the immune response should be considered at this point as adjuvants for vaccines, which, although they cause a strong activation of the cytotoxic lymphocytes, complicate the accumulation of the particles and the economic production thereof and are associated with an increased risk of toxicity and physiological transport restrictions.
WO 2001/12221 Al describes for silicon dioxide an intrinsic adjuvant effect in combination with proteinogenic antigens, cells or cell fragments, which is based on rough edges and an irregular shape, as a consequence of which penetration is facilitated of cell membranes and the modification of surface proteins. In contrast, WO 2007/030901 Al and Vallhov et al. (2007), Nano Lett. 7 (12), 3576, associate the adjuvant effect with the mesoporosity of the silica particles. Regardless of the fundamental cause, EP 0 465 081 Bl has already taught a preparation comprising a particle of metal core, ceramic (for example silicon dioxide) or polymer, a coating that covers at least partially the surface of this core particle, which comprises a basic sugar, a modified sugar or an oligonucleotide and at least one viral protein or peptide which is in contact with the coated core particle. The core particles have a diameter of 10 to 200 nm, but they agglomerate to form larger particles, which is still desired, since a deposition effect is thus established. It is disadvantageous that agglomerations of this type imply that it is not possible to produce pharmaceutically stable suspensions or achieve sterile filterability.
The invention is based on the objective of overcoming the disadvantages indicated in the prior art and developing nanoparticles which have a monodisperse particle size and make possible an effective application in immunoprophylaxis or immunotherapy, in particular as vaccines, which improve the therapeutic efficacy at the same time that they reduce the side effects.
The object of the invention is achieved in accordance with the independent claims. The sub-claims contain preferred embodiments. According to the invention, nanoparticles are provided which comprise a matrix which comprises more than 50% silicon dioxide, wherein the silicon dioxide has at least one surface functionality to which at least one antigen binds and where the nanoparticles have a size of 5 to 50 nm. The particle size must be interpreted at this point in such a way that a random distribution over the entire range between 5 and 50 nm is not present, but instead a defined particle size is selected within the range mentioned above, from which the deviation Standard is a maximum of 15%, preferably a maximum of 10%. In one embodiment of the present invention, the particles have a size between 10 and 30 nm, preferably between 20 and 30 nm, particularly preferably between 13 and 29 nm, very particularly preferably 25 nm ± 10%.
Surprisingly, it has been found that the provision of silicon dioxide nanoparticles in a reduced size range between 5 and 50 nm can significantly increase the effectiveness of antigen targeting in antigen presenting cells. In particular, the peripheral dendritic cells are no longer primarily targeted, but instead the dendritic cells of the lymph nodes. The nanoparticles according to the invention are designed through their size and the choice of materials in such a way that an effective induction of maturation of the dendritic cells takes place. This induction occurs, in particular, via the activation of the complement system. The nanoparticles of silicon dioxide according to the invention thus open up completely new opportunities with respect to the targeting of lymph nodes that have a high density of dendritic cells and with respect to the pathway of dendritic cell maturation as a pre-requisite for the proliferation and immunization of T cells. It is remarkable that a vaccine based on these nanoparticles does not need adjuvants which would otherwise be unavoidable in vaccination.
To date, it is only known from US 6,086,881 that the vaccine material must have a high molecular weight which increases the probability of antigenic determinants. Similarly, it is desired that the vaccine material be added or absorbed into alum or other gels since it usually becomes more effective with respect to binding to cells and stimulation of cell surface molecules and the antigen is retained in the tissue for longer periods due to the slow rate of desorption. It is also confirmed by Vallhov et al. (2007), Nano Lett. 7 (12), 3576, that the larger particles comprising mesoporous silicon dioxide have a greater influence on human dendritic cells that are derived from monocytes. In addition, antigen-silica conjugates for targeting in cells that present antigens, for which a particle size of 0.3 to 20 μ ?? it is considered as a necessary prerequisite for phagocytosis, they are described in the prior art according to WO 2008/019366 A2. In contrast, the present invention reveals that specifically nanoparticles of silicon dioxide in a range of reduced size, defined from 5 to 50 nm are capable of; Passive targeting in antigen-presenting cells and complement activation.
A "cell presenting antigens" in the sense of the invention is taken to refer to any cell which can be induced to present antigens for a T cell, which also includes precursor cells which are differentiated and activated for the cells that present antigens. Cells that present antigens include dendritic cells, Langerhans cells, PBMCs, macrophages, B lymphocytes or other types of activated or modified cells, such as, for example, epithelial cells, fibroblasts and endothelial cells which express MHC molecules on their surfaces cells, preferably dendritic cells, particularly preferably dendritic cells of the lymph nodes. The precursors of cells that present antigens include CD34 + cells, monocytes, fibroblasts and endothelial cells.
According to the invention, the particulate binding matrix comprises more than 50% silicon dioxide. The bond matrix can also be mixed in this way with additional components, where the silicon dioxide exhibits the highest ratio in a multi-component system. Examples of other components are metals, metal derivatives, metal oxides, polymers, organosilanes, other ceramics or glass. In an embodiment of the present invention, however, polymers are excluded as additional components. It is preferred that the matrix comprises at least 80% silicon dioxide, particularly preferably at least 90%. In a very particularly preferred embodiment of the nanoparticles according to the invention, the matrix comprises silicon dioxide which is essentially pure, ie it only comprises the impurities that are expected in the course of the preparation process. In an extremely preferred embodiment of the invention, the particulate bond matrix consists of silicon dioxide.
The particles can be prepared using, inter alia, the classic Stóber synthesis, in which silicon dioxide of monodisperity nanometric scale of defined size can be prepared by hydrolysis of tetrahydroxysilane (TEOS) in an aqueous-alcoholic medium. Ammoniacal (J. Colloid Interface Sci. 1968, 26, 62). Surprisingly, the inventors were able to show that the stability of the nanoparticles is retained despite the functionalization of the surface, as a consequence of which monodisperse particles are obtained which do not tend towards agglomeration. According to the present invention preference is given to nanoparticles produced by means of a process having the following steps: (a) hydrolytic polycondensation of tetraalkoxysilanes and / or organotrialkoxysilanes in a medium which comprises water, at least one solubilizer and at least one amine or ammonia, where first a primary particle sol is produced and the resulting nanoparticles are carried subsequent to the desired particle size in a range of 5 to 50 nm in such a way that further nucleation is prevented by means of the continuous input measurement of the corresponding silane in a controlled manner corresponding to the degree of reaction and (b) the binding of an antigen to a functionality of the surface of the nanoparticles.
If ammonia is a constituent of the medium, the solubilizer used is, in particular, alcohol, so that the reaction proceeds in an aqueous-alcoholic-ammoniacal medium, providing highly monodisperse particles whose standard deviation of the average particle diameter is not greater than 10% Surprisingly, the inventors have now discovered that the process still makes it possible for particle diameters of less than 50 nm to be achieved with the desired monodisperse properties. Step (a) of the process is described in detail in EP 0 216 278 Bl and WO 2005/085135 A1 and consequently these documents are incorporated in their entirety in the content of the description of the present invention by way of reference . At least one amine is preferably used in the medium.
The silicon dioxide matrix of the nanoparticles according to the invention can be either porous or non-porous. Porosity is essentially dependent on the production process. In the synthesis according to EP 0 216 278 Bl, in particular non-porous particles are obtained. Within the input range of 5 to 50 nm, a preferred particle size for the non-porous nanoparticles is between 10 and 30 nm, while the preferred particle size for the porous particles is 10 to 40 nm. The preferred particles of the invention are solid. j In connection with the present invention,! A "nanoparticle" is taken to refer to a particulate binding matrix which has functionalities on its surface which function as recognition points for the antigens to be linked or finally adsorbed. The Í The surface includes all the areas at this point, that is to say, in addition to the outer surface, also the interior surface of cavities (pores) in the particle. In an embodiment according to the invention, the antigen can be absorbed consequently into the particles, which requires porosity of the silicon dioxide matrix. ! The functionality of the surface may consist of one or more chemical groups, which may themselves be identical or different, where the groups either make possible the specific binding of nanoparticles and an antigen on their property as connectors or form a zeta potential not specific for the union. j The term "binding" here refers to any type of interaction between the functionality of the surface and the antigen, in particular covalent or non-covalent bonds, such as, for example, a covalent bond, hydrophobic / hydrophilic interactions, forces der Waals, ionic bond, hydrogen bonds, ligand-receptor interactions, nucleotide base pairing or interactions between a binding site of epitopes and antibodies. j In a preferred embodiment of the present invention, the antigen is covalently bound to the nanoparticle. The covalent bond can take place either indirectly. In the direct variant, the antigen directly on a chemical group in the particle, which usually takes place in a non-specific manner for the site and can make further release in the phagosome of the cell presenting antigens more difficult. In one embodiment of the invention, it is desired that the | thioethers, carbohydrates and / or oligonucleotides are excluded as surface functionality. The indirect method of covalent binding uses a connector or label via the I which antigen binds specifically to the site to the particles and is released again in a controlled manner. Labels for site-specific conjugation are known from the prior art, such as, for example, SNAP tag, halo tag, C-terminal LPX † G tag, biotin receptor peptide, PCP tag or yjbbR and are described , inter alia, in WO 2008/019366; A2 and consequently this document is incorporated in its entirety in the content of the description of the present invention by way of reference. This reference also has application with respect to all additional mentions of | this document in the course of this specification. ! In a preferred embodiment of the functionality of the surface, it is represented by means of a labile connector, particularly preferably by a hydrazone linker, disulfide linker or which is easily accessible from first clinical candidate, doxorubicin binds to the I polymer via an acid-labile hydrazone linkage as a nominal breakpoint (Angew, Chem. 2006, 118, 1218). i The macromolecules are absorbed into the cell by means of endocytosis, whereas a | drop I significant in the pH of the physiological value in space i t extracellular (pH 7.2 - 7.4) at pH 6.5 - 6 in the endosome and at pH I 4 in primary and secondary lysosomes. If the pH drops below 6 as a consequence of cellular uptake, the hydrazone bond breaks and the active compound is released by the polymeric support. The additional cleavable connectors which are suitable for the purposes of the invention and are described in the further course of the specification are known to the person skilled in the art. \ In a further preferred embodiment of the surface functionality, it is selected from the group of alkoxysilanes. It is particularly preferred at this point that it be a reactive, terminal thiol group. The alkoxysilanes can be used both for the binding of the antigen as well as for additional ligands of other functions, where the binding of the latter by means of this stable linker is preferred. The alkoxysilanes suitable for the purposes of the nanoparticles according to the invention can be selected routinely by the person skilled in the art.
In another embodiment of the present invention, the antigen is adsorbed on the nanoparticle. The adsorption can be carried out, for example, by mixing the antigen with the particles within a defined period of time, after which the nanoparticles are separated from the mixture, such as, for example, by means of centrifugation or filtration. The charge can still take place during the synthesis of particles. For the purposes of the invention it goes without saying that the adsorption also requires a suitable surface functionality (zeta potential), which can be either an inherent constituent of the matrix or has to be introduced I otherwise.; If the surface does not previously have a functionalization, depending on the synthetic route selected, it is introduced before the antigen binding. If the nanoparticles are produced by means of the hydrolytic polycondensation according to the above-mentioned process step (a), the functionalization of the surface is carried out after step (a) and before the i step (b). Many of the silicon atoms on the shell of the particle carry hydroxyl functions. Which are able to react with a multiplicity! of trialcoxysilanes or trichlorosilanes commercially available by means of standard methods, which means that the particles can be functionalized in various ways in a simple manner (J. Liq Chrom. &Techn.R. 1996, 19, 2723).
If the desired applications or desired properties of nanoscale silicon dioxide particles require greater chemical complexity, well-structured, multi-step synthesis is used. j Finally, the antigen binds to the nanoparticle by means of interaction with the functionality of the surface. i An "antigen" is taken at this point to refer to a structure which is capable of generating a cellular or animal immune response. It goes without saying that the immune response in an animal includes all mammals, particularly humans. The antigens are preferably proteinogenic, ie they are proteins, polypeptides, peptides or fragments thereof, which in turn can be of any desired size, origin and molecular weight and can; be 1 glycosylated, but contain at least one antigenic determinant or an antigenic epitope. Recognition through the immune system takes place, in particular, from a minimum length of three amino acids. The proteins or peptides are preferably selected from the group of cytokines, receptors, lectins, avidins, lipoproteins, glycoproteins, oligopeptides, peptide ligands and peptide hormones. The antigens can also be nucleic acids per se or encoded by nucleic acids, which, after transport within the nucleus of antigen presenting cells, are translated into the proteinogen antigen which is presented to the MHC molecules. The nucleic acids are single and double stranded DNA or RNA and oligonucleotides. The nucleic acids can also be a constituent of complexes or formulations which consist of livides, carbohydrates, proteins or peptides. The additional antigens are polysaccharides, polymers, low molecular weight substances having a molecular weight of 50 to 1000 Da, viruses, intact prokaryotic or eukaryotic cells or cell fragments.
I In one embodiment of the invention, the antigen ¡has a molecular weight less than 500 kDa. The antigen is preferably a cancer antigen. Cancer antigens of this type are disclosed, for example, in WO 2008/019366 A2. In a particularly preferred embodiment, the cancer antigen is selected from the group consisting of the New York esophageal antigen (NY-ESO-I), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6 , MAGE-A8, MAGE-A10, MAGE-B, MAGE-C1, MAGE-C2, L antigen (LAGE), SSX2, SSX4, SSX5, PRAME, melan-A, caspase-8, tyrosinase, MAGF, PSA, 'CEA, HER2 / neu, MUC-1, MARTI, BCR-abl ,. p53, ras, myc, RB-1 and i survivin or epitopes thereof. In a very modality I particularly preferred of the invention, the antigen of the I Cancer is survivin or epitopes of it. This cancer antigen is described in WO 2007/039192 j A2 and consequently this document is incorporated in its entirety in the content of the description of the present invention by way of reference. ' In another embodiment of the invention, MHC receptors and / or molecules are excluded as surface functionality and as antigens.
The nanoparticles can be multifunctionalized, which means, in the sense of the invention, different chemical groups (functionalities of the surface) and / or different linked molecules (functions). It is preferred that both the functionalities of the surface and the linked functions are different, giving rise to an independent link, specific to the functional molecules. The functions are preferably selected from the group of antigen, polyethylene glycol (PEG), labeling and adjuvant, where it goes without saying that the antigen is always selected. The antigen and PEG and / or adjuvant are present in a particularly preferable manner; very particularly preferable is the antigen, PEG and adjuvant, where these functions can be linked in an adsorbent and / or covalent manner.
In one embodiment of the particles according to the invention, the labeling is detected by means of the luminescence, UV / VIS staining in an enzymatic, electrochemical or radioactive manner. Fluorescent dyes or radioactive labels are preferably used. In the case of photoluminescence or fluorescence, the excitation is carried out by means of the absorption of photons. The preferred fluorophores are bisbenzimidazoles, fluorescein, Acridine orange, Cy5, Cy3 or propidium iodide. The I I The evaluation is carried out visually or using appropriate measuring instruments, for example under a fluorescence microscope or by means of flow cytometry, for example in a cytofluorimeter. In a particularly preferred embodiment of the invention, the fluorescent dyes bind to 3-aminopropyltriethoxysilane, where the fluorescein isothiocyanate is a very particularly preferred fluorescent dye.
Alternatively, the detection can also be I i carry out radioactively using radioactive isotopes, preferably using 3H, 14C, 32P, 33P ', 35S, 99mTc, 11 ??? or 1 5I, particularly preferably using 99mTc or 11: LIn. In particular, derivatives of 1,4,7,10-tetraazacyclododecane-N, N ', N "N"' - tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA), which bind to nanoparticles via Click or modular chemistry is provided with the particularly preferred radioisotopes immediately prior to injection. In case i of the scintillation count, a cocktail of molecules is excited, for example, by means of radioactive radiation? The energy released as light in the transition in the ground state is i amplified by a photoelectron multiplier and it is counted.
In this way, the nanoparticles according to the and invention are also important as a diagnostic tool (for example in imaging methods) and / or research tool, which makes it possible to visualize the fixation as an objective and the uptake of the active compound.; In a further embodiment of the invention, an antigen is combined with a tag such that the assignment to the antigen can be carried out within a particle fraction via the tag. This means that a first particle or a plurality thereof is i provided with a first antigen and a first label, while a second particle or a plurality thereof is provided with a second antigen and a second one I label, etc., where both the antigens and the labels are in each case different from each other. Consequently, the specific combination of antigen and label is unique and is preferred in this document and | it makes possible the mixing of particles with different antigens and the parallel supervision of the fixing efficiency; as objective and / or immune activation / complement. This results in a saving of time in the diagnoses in comparison with the sequential administration. Of course, in the same way it is possible for the particles to carry a plurality of antigens and a plurality of labels 1 whose intensities vary, which means that a certain antigen can be selected from the mixture. The label is preferably a fluorescent dye, which is linked, in particular, to silane.
Additionally, the nanoparticles of the invention can be designed as combinations of antigens or danger signals, such as, for example, TLRs or cytokines.
In still a further embodiment of the present I invention, the surface is multifunctionalized in such a way that the cross-linking of the multifunctionalities is excluded. t The previous teaching of the invention and the I I modalities thereof which relate to the functionality of the surface for the binding of antigens to nanoparticles are valid for and applicable without restriction to the multifunctionalities and / or the binding of additional functions to j nanoparticles, as long as it seems appropriate. j.
A universal strategy for the construction of highly complex systems is the concept of chemistry, click or modular presented by K. B. Sharpless (Angew.Chem.j Int.
Ed. 2001, 40, 2004). This is more of a philosophy of synthesis than a scientific discipline, which is inspired, in particular, by the simplicity and efficiency of reactions of natural origin. A fundamental example of chemical or modular chemistry has proven to be the 1,3-dipolar cycloaddition of azides and terminal alkynes by means of the Huisgeri method. In the presence of monovalent copper, these reactions take place with a drastic acceleration and also proceed regioselectively, in very high yields and with tolerance of a wide range of functional groups. An additional advantage is found in the possibility of carrying out the synthesis in an aqueous medium and at room temperature, making it possible for the interesting biomolecules to be joined together I Modular way and widely applicable to other building blocks in a type of established construction principle. Therefore, for the purposes of the invention it is preferred to join the dioxide particles of í. silicon functionalized correspondingly to the functions mentioned above, in particular the antigens, using click or modular chemistry.
The invention also relates to a dispersion the Which comprises the nanoparticles according to the invention. The nanoparticles can be in dispersed form in any desired solvent, as long as | the nanoparticles are not chemically attacked or physically modified by the solvent, and vice versa, so that the resulting nanodispersion is stable, particularly pharmaceutically and physically stable. The dispersion is characterized specifically because the nanoparticles are in a monodisperse and non-aggregated form and have no tendency towards sedimentation, which results in sterile filterability. The previous teaching of the invention and the modalities of it that refer to | the ! Nanoparticles are valid for and applicable without restrictions to dispersions, as long as it seems appropriate.
I The invention can also be practiced as a kit which comprises the nanoparticles according to the invention and / or dispersions thereof, the kit of the invention can also contain an article which contains written instructions or indicates to the user written instructions which explain the handling of the nanoparticles of the invention. The previous teaching of the invention and the The modalities of the kit that refer to the nanoparticles and their dispersions are valid for and applicable without restrictions to the kit, as long as it seems appropriate.
The invention also relates to a pharmaceutical composition which comprises the nanoparticles according to the invention or dispersions thereof. A "pharmaceutical composition" at this point is any composition which can be used in the prophylaxis, therapy, control or post-treatment of patients who exhibit, at least temporarily, a pathogenic modification of the complete condition or the condition of individual parts of the organism. of the patient, particularly as a result of i infectious diseases, septic shock, tumors, cancer, I autoimmune diseases, allergies and chronic or acute inflammation processes. Thus, in particular, it is possible for the pharmaceutical composition in the sense of the invention to be a vaccine and / or an immunotherapeutic agent. The pharmaceutical composition may comprise antigens, such as, for example, peptides or nucleic acids, for example, as a pharmaceutically acceptable salt. This may be, inter alia, salts of inorganic acids, such as, for example, phosphoric acid or salts of organic acids.
In order to withstand the medical effect, that is, in i | particular, the immune response. The pharmaceutical composition can also, in one embodiment of the invention, comprise additional active compounds, where simultaneous or successive administration is conceivable. The therapeutic effect of the pharmaceutical composition according to the invention may arise, for example, through certain antitumor drugs that have a better action through the activation of the complement system as a desired side effect or through the variety of effects collateral of those medications that are reduced by reducing the dose.
In a preferred embodiment of the invention, the pharmaceutical composition according to the invention is combined with chemotherapeutic agents, which are I select from the group comprising cytokines, chemokines, I pro-apoptotic agents, interferons, radioactive compounds or combinations thereof. It is preferred that the chemotherapeutic agent modify, in particular reduce! the metabolism of nucleic acids and / or proteins, cellular resignation, DNA replication, purine biosynthesis, pyrimidine and / or amino acids, gene expression, mRNA processing, protein synthesis, apoptosis or combinations thereof. i I In order to stimulate the endogenous defenses of strengthening the immune system, it is also possible, in a further embodiment of the invention, to administer immunostimulants, for example interferons, such as, for example, IFN-α, IFN-β or IFN- ?, Interleukins, such as, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 or IL-12, tumor necrosis factors, such as], for example, TNF-α or TNF-β, erythropoietin, M-CSF, G-CSF, GM-CSF, CD2 or ICAM, with the present pharmaceutical composition. In this way, the proliferation, development, differentiation or activation of, for example, T lymphocytes can be stimulated, B lymphocytes, monocytes, macrophages, neutrophil cells, eosinophil cells, megakaryocytes and / or granulocytes. I In order to increase the protective or therapeutic action of the immunogenic nanoparticles according to the invention, the pharmaceutically tolerated adjuvants can be added to the particles or all the compositions I pharmaceutical preparations prepared therefrom. For the purposes of the invention, any substance which facilitates, ameliorates or modifies an effect with the antigens according to the invention is an "adjuvant". Known adjuvants are, for example, aluminum compounds, such as, for example, aluminum or aluminum phosphate, saponins, such as, for example, QS 21, muramyl dipeptide or muramyl tripeptide, proteins, such as, for example, gamma-interferon or TNF, MF 59, phosphatidylcholine, squalene or polyols. The co-application of egg albumin in Freund's complete adjuvant can cause the same: mode an increased cell-mediated immunity and. this ? way it can withstand the action of neutralizing antibodies formed. Additionally, DNA, | which has an immunostimulatory property, or which encodes a protein with an adjuvant effect, such as, for example, a cytokine, can be applied in parallel or in a construct. However, due to the intrinsic adjuvant effect of the nanoparticles based on silicon dioxide according to the invention, it is preferred in the present case not to use i additional adjuvants. If the intrinsic adjuvant effect is inadequate in certain applications, it is of course possible to additionally bind one or more adjuvants to the nanoparticles, preferably only one adjuvant. The binding ring may be either absorbent or may be a covalent bond. The preferred adjuvants of the invention to I to be bound in an absorbent manner include poloxamers and 1 I TLRs. The covalently bonded adjuvants, preferred and the invention include short chain peptides, particularly preferably tuftsin or ovalbumin.
The introduction of the pharmaceutical composition into a cell or an organism can be carried out in accordance I with the invention in any way which makes it possible for I the cells that present antigens are put in contact with the nanoparticles or antigens present in the composition and are absorbed in the cells by means of phagocytosis, as a consequence of which an immune response is induced.
The pharmaceutical composition of the present invention can be administered by the oral, transdermal, transmucosal, transurethral, vaginal, rectal, pulmonary, enteric, and / or parenteral route. The parenteral administration of the pharmaceutical composition is preferred. It has been shown in the present1 case that the silicon dioxide on its property as an adjuvant does not have adverse effects on the lipid balance, as it is observed for the polymeric adjuvants, which are not I Approved accordingly for this type of application. Direct injection into the body is particularly preferred. He I The type of administration selected depends on the indication, the dose to be administered, specific parameters of the individual, and so on. In particular, the various types of administration facilitate site-specific therapy, which minimizes side effects and reduces the dose of active compound. Very particularly preferred injections are intradermal, subcutaneous, intramuscular or intravenous injection. The administration can be carried out, for example, with the aid of the so-called vaccination guns or by means of syringes. It is also possible to prepare the substance as an aerosol, which is inhaled by the body, preferably a human patient.
The forms of administration of the composition I Pharmaceutical preparations are prepared according to the type of administration desired in a suitable dosage and in a manner known per se using conventional solid or liquid vehicles and / or diluents and the auxiliaries usually employed. In this way, pharmaceutically acceptable excipients that are known to the person skilled in the art can basically form part of the pharmaceutical composition according to the invention, wherein the amount of excipient material that is combined with the active compound in order to preparing an individual dose varies depending on the individual being treated and the type of administration. These pharmaceutically tolerated additives include salts, buffers, fillers, stabilizers, and complexing agents before, antioxidants, solvents, binding agents, lubricants, tablet coatings, flavors, dyes, preservatives, adjusters and the like; Examples of excipients of this type are water, vegetable oils, benzyl alcohols, alkylene glycol, polyethylene glycol, glycerol triacetate, gelatin, carbohydrates, such as, for example, lactose or starch, magnesium stearate, talc and Vaseline.
The pharmaceutical formulation may be in the form of a tablet, film-coated tablet, dragee, lozenge, capsule, pill, powder, granules, syrup, juice, drops, solution, dispersion, suspension, suppository, emulsion, implant, cream, gel, ointment, paste, lotion, serum, oil, spray, spray, adhesive, plaster or bandage, where dispersion is preferred.
The oral administration forms that are prepared are preferably tablets, film coated tablets, dragees, rhombic lozenges, capsules, powder pills, granules, syrups, juices, drops, solutions, dispersions or suspensions - including as a reservoir form. The forms of drugs such as tablets can be obtained, for example, by mixing the activated compound with known auxiliaries, such as dextrose, sugar, sorbitol, mannitol, polyvinylpyrrolidone, disintegrants, such; such as corn starch or alginic acid, binding agents, such as starch or gelatin, lubricants, such as magnesium stearate or talc and / or agents which are capable of achieving a deposition effect, such as carboxypolymethylene, carboxymethylcellulose, phthalate cellulose acetate or polyvinyl acetate. The tablets may also consist of a plurality of layers. Dragees can be prepared in the same way by coating cores produced analogously to the tablets with agents usually used in dragee coatings, for example, polyvinylpyrrolidone or shellac, gum arabic, talcum, titanium dioxide or sugar. The coating of the dragee at this point may also consist of a plurality of layers, where, for example, the aforementioned auxiliaries are used. Capsules can be produced by mixing the active compound with vehicles, such as lactose or sorbitol, which are then filled into capsules. The solutions or dispersions of the pharmaceutical composition can be mixed with substances, such as, for example, saccharin, cyclamate or sugar types and / or with flavors, such as, for example, vanilla or orange extract, I to improve the flavor. Additionally, they can be mixed with suspension aids, such as, for example, sodium carboxymethylcellulose or preservatives, such as, for example, p-hydroxybenzoic acid, phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, benzaikonium chloride or benzethonium chloride. ! ..i Additionally, the forms of medications t parenteral, such as, for example, suppositories, suspensions, emulsions, implants or solutions should be considered, preferably oily or aqueous solutions. For parenteral administration, the immunogenic construct of the invention can be dissolved or suspended in a physiologically tolerated diluent, such as, for example, neutral fats or polyethylene glycols or derivatives thereof. Preferred solvents which are used are frequently oils, with or without the addition of a solubilizer, a surfactant, a suspending agent or an emulsifier. Examples of oils used are olive oil, peanut oil, cottonseed oil, castor oil and I sesame oil. | For topical application of the pharmaceutical composition, the latter is formulated in a conventional manner with at least one pharmaceutically acceptable carrier, such as, for example, microcrystalline cellulose and optionally additional auxiliaries, such as, for example, moisturizers, to provide formulations solid which can be applied to the skin, such as, for example, creams, gels, ointments, pastes, powders or emulsions, or to provide liquid formulations which can be applied to the skin, such as, for example, solutions, suspensions, lotions, serums, oils, sprays or aerosols. Examples are solutions in alcohols, such as, for example, ethanol or isopropanol, acetonitrile, DMF, dimethylacetamide, 1,2-propanediol or mixtures thereof with each other and / or with water. The liposomes, which guarantee the Optimal transport within the skin, they can also serve as carrier systems for the pharmaceutical composition. The they can comprise pharmaceutically acceptable absorbable solvents in order to withstand the passage of the nanoparticles through the skin. The solvents which guarantee a good penetration into the skin are, for example, the alcohols phenyl-1-ethanol, glycerol, ethanol or mixtures thereof.
The pharmaceutical composition is preferably in the form of a solution for injection. For the preparation of the solution for injection, means may be used and aqueous, such as, for example, distilled water or physiological salt solutions, where the latter include acid and basic addition salts. The pharmaceutical composition can also be in the form of a solid composition, for example in the lyophilized state, and then it can be prepared before use by the addition of a dissolving agent, such as, for example, distilled water. The expert person in the field is familiar with the basic principles and the preparation of lyophilizates.
The concentration of active nanoparticles; in the formulation it can vary between 0.1 to 100 percent in weight. It is crucial that the pharmaceutical composition understand! as an active compound, an effective amount of the nanoparticles and / or a dispersion thereof together with the pharmaceutically tolerated auxiliaries. The terms "effective amount" or "effective dose" are used interchangeably in I this document and indicates an amount of the pharmaceutically active compound which has a prophylactic or therapeutically relevant action on a disease or pathological change. A "prophylactic action" prevents the beginning of ? a disease or even infection with a pathogen after the entry of individual representatives in such a way that the subsequent propagation of the same is reduced in: large measure or even completely deactivated. A "therapeutically relevant action" releases one or more symptoms of disease or results in partial or complete reversal of one or more physiological or biochemical parameters which are associated with or causally involved in the disease or pathological change, in the normal. The jdosis 1 or respective dose range for the administration of the nanoparticles according to the invention is sufficiently I large to achieve the desired prophylactic or therapeutic effect of the induction of an immune response. In general, the dose will vary with the age, constitution and gender of the patient and the severity of the disease will be taken into account. Needless to say, the specific dose, frequency and duration of the administration are, in addition, dependent on a multiplicity of factors, such as, for example, the binding capacity as an objective and binding of the nanoparticles, the individual's nutrition habits. it is treated, type of administration, excretion rate and combination with other medications. The individual dose can be adjusted both with respect to the primary disease and also with respect to the appearance of any complication. The precise dose may be established by a person skilled in the art using known means and methods. This teaching of the invention is valid for and applicable without restrictions to the pharmaceutical composition comprising the nanoparticles and / or dispersions thereof, serier and when it seems appropriate. i In one embodiment of the invention, the nanoparticles are administered in a dose of 0.01 mg to 1 g per kilogram of body weight per day. However, doses of 20 to 60 mg per kilogram of body weight per day are preferably administered. The daily dose is preferably between 0. 02 and 10 mg / kg of body weight. j, According to the invention, the present nanoparticles and / or nanoparticle dispersions j are suitable for the prophylactic or therapeutic treatment of diseases which are selected from the group of infectious diseases, septic shock, tumors, cancer, autoimmune diseases, allergies and processes of chronic or acute inflammation. It goes without saying that the host of the pharmaceutical composition is also included in the scope of protection of the present invention. j In a preferred embodiment, the cancer or tumor disease that is treated, prophylactically prevented or whose recurrence is prevented is selected from the group of cancerous or tumoral diseases of the otorhinolaryngological region, mediastinal cavity, gastrointestinal racti (including: colon carcinomas, carcinomas stomach, colon cancer, small bowel cancer, pancreatic carcinomas, hepatic carcinomas), urogenital system (including renal cell carcinomas), gynecological system (including ovarian carcinomas) and endocrine system and lungs (including lung cancer), 1 breast (including breast carcinomas) and skin and bones and soft-tissue sarcomas, mesotheliomas, melanomas, neoplasms of the central nervous system, cancerous diseases or pediatric tumor diseases, lympholas, leukemias, paraneoplastic syndromes, metastases without known primary tumor I (CUP syndrome), peritoneal carcinomatosis, malignancies related to immunosuppression, multiple myelomas and tumor metastasis.
The autoimmune diseases to which the invention relates are preferably selected from the group comprising arthritis, autoimmune hepatitis, chronic gastritis, neurodermatitis, psoriasis, osteoarthritis, rheumatic diseases, reutomatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, festering inflammation of the colon, diabetes, inflammatory bowel diseases, multiple sclerosis and / or allergic inflammations. i According to the invention, the nanoparticles j are also used for the prophylaxis or therapy of diseases which are caused by microorganisms which can be pathogenic for mammals. This means that the action according to the invention is directed against either microorganisms which are capable of carrying out processes that damage health for their own benefit through an alteration in the natural balance of the microflora which colonizes an organism. host and / or in the case of hosts which have a weakened immune system, or against those which are inherently pathog). Preferred microorganisms in the sense of the invention are viruses, bacteria, fungi and / or unicellular animals. Particular preference is given to bacteria, where Gram-positive and Gram-negative bacteria are influenced in their growth. Examples of diseases which can be treated with nanoparticles are hepatitis B, hepitis C, HIV, herpes, tuberculosis, leprosy or malaria, which are caused by the microorganisms mentioned above.
It is known to the person skilled in the art that the induction of T cell proliferation and / or neutralizing antibodies can be advantageous at virtually any time. In the present case, the nanoparticles and °! : dispersions of them are used mainly for immunotherapy, which means that vaccination in the I The invention is preferably an administration of the pharmaceutical composition according to the invention after diagnosis and / or onset of a disease which responds to immunotherapy. Vaccination should preferably be carried out shortly after the diagnosis or onset of the disease and it can also be administered a variety of times as therapy in order to improve the initial, proliferative, immune response of the organism by a variety of injections. Consequently, the supervision is also > It takes to imply a type of therapeutic treatment if the nanoparticles are administered at certain intervals! for example in order to completely eliminate the symptoms of a disease. In a preferred embodiment of the present I invention, the nanoparticles and / or dispersions thereof are used for the therapy of cancer and / or tumors, particularly preferably for cancer therapy.
Naturally, it is advantageously possible in the same way for active vaccination to develop protection after prophylactic administration in the organism. Prophylactic immunotherapy is advisable, in particular, if an individual is predisposed to the onset of the diseases mentioned above, such as, for example, a family history, a genetic defect or a recently overcome disease.
In this way, the invention also relates to the use of the nanoparticles according to the invention and / or the dispersion according to the invention. immunoprophylaxis or immunotherapy. The invention also relates to the use of an effective amount of the nanoparticles according to the invention and / or the dispersion according to the invention for the preparation of a vaccine for immunoprophylaxis or immunotherapy. In both subjects, the diseases to be treated are selected from the group: which includes infectious diseases, septic shock, tumors, »cancer, autoimmune diseases, allergies and cancer processes.
I chronic or acute inflammation. The vaccine is prepared, in particular, by means of non-chemical methods by converting the active compound into a suitable dosage form point with at least one vehicle or solid auxiliary, liquid and / or semi-liquid and optionally in combination with one? more additional active compounds. The previous teaching of the invention and its modalities are valid for and applicable without restrictions to the nanoparticles, dispersions and the medical use thereof, as long as it seems appropriate. \ A further embodiment of the invention relates to the use of the nanoparticles according to the invention and / or dispersions thereof for the targeting of antigens in antigen presenting cells and optionally for activation of the immune system, preferably for activation of the complement system. The targeting is preferably carried out ex vivo or in vitro by administering the nanoparticles carrying antigens to the cells, cell cultures, tissues or organs which comprise antigen presenting cells. Ex vivo use is used, in particular, in the case of animal cells.
I ! which originate from an animal organism which is affected by a disease selected from the group of infectious diseases, septic shock, tumors, cancer, autoimmune diseases, allergies and chronic or acute inflammation processes. Cells treated ex vivo may either continue to be cultured for subsequent investigations or may be transferred into an animal, which may be the host animal or another animal. Filing as an ex vivo target according to the invention is advantageous, in particular, in order to test the specific structure of the nanoparticles with respect to particle size, antigen, binding and multifunctionalization, making it possible for the in vivo dose to be established i correspondingly before the evaluation of this ex vivo data. As a result, the therapeutic effect on the form of the acquired immune response is significantly increased. In the same way, it is possible to stimulate the T cells of a patient outside the body directly by means of the cells that present antigens which were exposed to the nanoparticles and then either implant the cells! T or use T cells for research purposes.
In a preferred embodiment of the use according to the invention, the antigens are directed to dendritic cells. In a particularly preferred embodiment of this use, the dendritic cells are located in the lymph nodes. It goes without saying that the last mentioned modality requires at least one tissue or organ, but in I at best, an intact animal organism. Similarly, it goes without saying that this prerequisite; must be satisfied for immune activation or especially complement activation. j i Nanoparticles can be used,! consequently, in vivo by administering them directly to an animal, in particular a mammal, particularly preferably a human, via known routes; The nanoparticles can be used additionally ex vivo, where I the antigen-presenting cells are first isolated from an animal and subsequently treated ex vivo with the nanoparticles according to the invention in such a way that the nanoparticles are absorbed by the cells. The cells that present antigens treated in this way are returned to the body, as a result of which the cells of the body are stimulated.
Accordingly, the invention further relates to a method for the targeting of antigens in antigen presenting cells having the following steps: (a) the provision of nanoparticles comprising essentially pure silicon dioxide which has a surface functionality to which at least one antigen binds; (b) the administration of the nanoparticles to cells that present antigens which are present in a cell culture, tissue, organs or an animal, (c) the targeting of the antigens in cells that present antigens via the interstitial fjluid adjust the efficiency of fixation as an objective via the size of the nanoparticles, which are at least partly inversely proportional.
I In step (a) of the method according to the invention, the nanoparticles are preferably provided by means of the following steps: (a ') the hydrolytic polycondensation of tetraalkoxysilanes and / or organotrialkoxysilanes in a medium which comprises water, at least one solubilizer and at least one amine or ammonia, where first a sojl of primary particles and nanoparticles is produced. resulting results are subsequently carried to the size of I desired particle in such a way that additional nucleation is prevented by means of the corresponding continuous silane input measurement in a controlled manner corresponding to the degree of reaction. (a ") the binding of at least one antigen to a surface functionality of the nanoparticles, and optionally (a "') the dispersion of the nanoparticles.
In step (b) of the method according to the i, invention, the nanoparticles are preferably administered to an animal, particularly preferably a mammal, very particularly preferably a human The administration is carried out, in particular, by the route parenterally, particularly preferably by | intradermal or subcutaneous route.
In step (c), it has been unexpectedly discovered that the targeting of the silicon dioxide particles can be influenced by the size of the nanoparticles. While a particle size of at approximately 150 nm represents the upper limit at which fixation is still observed as an objective, the efficiency of the fixation as an objective increases in a smaller particle size. The size range of the i particles is preferably greater than 0 nm and smaller than 150 nm, particularly preferably between 5 | and 50 I nm, very particularly preferably it is between 10 | and 30 nm much more preferably is between 13 and 29 nm. ! In one embodiment of the invention, the targeting efficiency and the size of the nanoparticles are inversely proportional throughout the range J The efficiency can be increased either in a linear or I. in a non-linear manner, preferably in a non-linear manner. ? i In another embodiment of the invention it is possible that the inverse proportionality between the fixing efficiency; as objective and particle size do not exist throughout the size range, but instead that the correlation according to the invention approaches a maximum value for the fixing efficiency as an objective that is not observed in the size of the smaller particle and in this way the end point of the size range. In this embodiment of the method according to the invention, the dependence of the fixation efficiency as an objective (in the ordinate) on the particle size (in the abscissa) is preferably described by an exponential function with regular, natural exponents greater that / equal to 2, whose parable is open in the I background, so that the vertex represents the maximum efficiency. A quadratic function is particularly preferred. In other words, this means that the inverse proportionality is observed for a vertex (inflection point) in the aforementioned particle size range between 0: and 150 nm. j By means of the method, the fixation as a partial objective can be specifically established or the enhancement of the fixation as an objective can be achieved. In one embodiment of the present method, more than 50%, preferably more than 70%, particularly preferably more than 85 percent, of I very particularly preferable more than 95%, give the cells that present antigens in the lymph nodes are targeted. For this purpose, it is further preferred to employ nanoparticles having a size of 5 to 50 nm. The particle size includes at least the silicon dioxide matrix, preferably the complete nanoparticle.
In a further embodiment of the method according to the invention, step (c) is followed by the additional steps: i (d) the absorption of the nanoparticles in the cells that present antigens and optionally i (e) the release of the antigens in the endosome.
It is preferred that both steps (d) and (e) be carried out after step (c). The kinetics of the release of antigens by the particulate binding matrix after idecytosis can be controlled in step (e) by the I antigens that are covalently linked to the vehicle via a commonly called cleavable linker. For example, a pH-sensitive linkage, an enzymatic interface (eg, a protease-sensitive linker) and / or a reductive or oxidatively cleavable linker may be incorporated | as surface functionality. Preferred pH-sensitive bonds of the invention are achieved by means of certain esters, disulfide linkages, a hydrazone linker, an anhydride link, self-cleavable intein sequences, pH-sensitive complexing agents or polymers, i such as, for example, γ-ß-amino-esters modified with polyethylene oxide. The covalent bonding of antigens via a labile linker as surface functionality is essential for step (e), where a hydrazone linker, a disulfide linker or a peptide sequence is preferred which is easily accessible in a manner enzymatic In addition, it is preferred that the antigens be released in the early endosome.
The previous teaching of the invention and modalities I of the same that refer to the nanoparticles, dispersions thereof, pharmaceutical compositions and use are valid for and applicable without restrictions to the method for targeting antigens in cells that present antigens, as long as it seems appropriate. j i The invention further relates to a method for the activation of the immune system in a mammal, in which, in S a first step (a), the nanoparticles are directed to cells that present antigens according to the method according to the invention described above and, in a second step i (b), the immune system is activated. The complement system is I i preferably active. In step (b), the activation efficiency can be adjusted via the characteristics of the particles, which include in particular the particle size, surface functionality, loading and type of I surface, ratio, amount and density of the ligandsj (for example antigen, PEG, adjuvant). It is preferred in pasp (b) to adjust the activation efficiency via the particle size, which are at least partly inversely proportional. The activation efficiency increases, in particular, with a smaller particle size. The previous teaching of the invention and modalities of the same which refer to the method for the targeting of antigens in cells presenting antigens is valid fara and applicable without restrictions to the method for the activation of the immune or complement system in a mammal , always and I when it seems appropriate.
The invention further teaches a method of vaccination, in which an effective amount of the nanoparticles according to the invention and / or dispersions comprising, these nanoparticles is administered to a mammal which | this I in need of this treatment. The mammal to be treated is preferably a human. The previous teaching of the I invention and modalities thereof are valid and applicable without restrictions to the method of treatment, as long as it seems appropriate.
The invention also teaches a method for the induction of a T cell response, antibody response and / or maturation of dendritic cells, characterized in that the nanoparticles according to the invention, which may be in the form of a dispersion and / or Pharmaceutical composition, a mammal is administered and the proliferation of T cells and / or dendritic cells and / or the formation of neutralizing antibodies is induced. Preferred organisms in the sense of the invention are human or animal. The description of the nanoparticles of I According to the invention, it is possible for the person skilled in the art to use these for the induction of T cells and / or neutralizing antibodies. 1 It is known to the person skilled in the art that he is capable of administering the nanoparticles according to the invention, which of course can also be used as a pharmaceutical composition according to the invention, in various dosages to an organism, in particular a patient human. The administration at this point will; it must be carried out in such a way that the largest possible amount of T cells and / or neutralizing antibodies is generated. The concentration and type of administration can be determined by the person skilled in the field by means of routine experiments. j i G The contact of the nanoparticles or the pharmaceutical composition can be carried out prophylactically or therapeutically. In the case of, for example, vaccination Prophylactic for the development of vaccination protection I active against viral infectious diseases, infection with viruses must be prevented at least in such a way that, after the entry of individual viruses,! by I example in a wound, the additional multiplication of them is greatly reduced or that the viruses that have entered are virtually eliminated completely. In the case of the therapeutic induction of an immune response, an infection of the patient already exists and the induction of the cells i T and / or neutralizing antibodies is carried out in order to eliminate the viruses that are already present in the body or to inhibit its multiplication. | i The invention also relates to a method for the passive immunization of an organism, characterized in that the T cells and / or antibodies which have been induced by the administration of the nanoparticles according to the and invention with a mammal are isolated and administered! to an additional mammal. An "additional mammal" in the sense of the invention is taken to refer to both organisms of the same species or of different species, but not the same organism which has induced the T cells and / or antibodies. It is also possible to isolate monoclonal antibodies, which are used, inter alia, after the corresponding humanization. The antibody producing cells can be isolated in the same way from vaccinated or infected individuals who produce neutralizing antibodies which are directed against the nanoparticles according to the invention and are administered in the form of monoclonal antibodies in the case of passive immunization. · In passive immunization, essentially no immune reaction inherent to, for example, certain viruses) takes place in the patient's body, but instead T cells I and / or antibodies are introduced into the patient, for example in the form of curative sera. In contrast to active immunization, passive immunization has the task of curing a ? infection that has already taken place as quickly as possible or alternatively to provide protection I immediately against a virus infection. Various schemes I of vaccination for passive immunization are known to the person skilled in the art, for example of passive immunization against hepatitis A, hepatitis B or FSME. Vaccination schemes of this type can be adapted to specific retroviruses, such as, for example, HIV, feline and leukemia viruses and others by means of routine experiments. The antibodies which are used for passive immunization are preferably monoclonal antibodies. These are used, in particular, as a constituent for therapy i I of combination.
I j ' All known and additional constituents or components are known to the person skilled in the art and can be subjected to a specific refinement for teaching according to the invention in experiments of í routine. I I I Within the structure of the present invention, a ! Ultra-small conjugate of silicon dioxide-antigen! which promotes an immune, cellular, effective response after vaccination is provided in this way for the first time. The conjugate is directed to a double action mechanism because it is capable of both binding and specific targeting in cells that present antigens as well as of simultaneous complement. The nanoparticles smaller than 50 nm have a fixation efficiency as the target! which is a higher multiple compared to the large nanoparticles of the prior art. As a consequence of the I efficient transfer within the lymphatic vessels, the biophysical mechanism of interstitial flow can be used i advantageously for fixing as target cells i dendritic lymph nodes. The convection of the nanoparticles in this new transport route makes fixation a passive target, as a consequence of which the fixation as a specific objective of complex cells is superfluous, but nevertheless a particularly large number of cells are reached since dendritic cells are present in a large number in the lymph nodes. These properties form the basis for the reliable recognition of lymphatic nodule dendritic cells - which includes the absence of cross-reactivities (including targeting of peripheral dendritic cells) - and reproducible, reliable and complete phagocytosis in these cells that present antigens. The second advantageous property of nanoparticles based on silicon dioxide, whose intrinsic adjuvant effect activates the immune system and in particular the complement system, has an effect on the target. While the potency of the activation is independent of the selected antigen, it can be modified via the particle size. The absence of auxiliaries and / or additional modifications of the surface of the nanoparticles (for example polyhydroxylation) for the activation of the immune or complement system represents a simplification and reduction of significant expenses.
The nanoparticles according to the invention are characterized by the inert, inert, biocompatible matrix material, which can be used, in particular, for prophylactic or therapeutic vaccination. The development of the nanoparticles comprising the silicon dioxide / antigen conjugate presented at this point is likewise an extremely promising strategy for improving the therapeutic index of cytotoxic active compounds. In particular, the labile binding of the constituents ensures the release of the antigenic therapeutic agent in specific compartments of the body, which means that. A reduction in possible side effects can be expected. Nanoparticles are also distinguished by high pharmaceutical stability and are easy to handle, at least because of their small size. Ultrafine nanodispersions comprising monodisperse sized particles are advantageously suitable for sterile filterability.
It goes without saying that this invention is not restricted to the specific methods, particles and conditions described in this document, since these things may vary. Furthermore, it goes without saying that the terminology used at this point serves exclusively for the purpose of describing particular embodiments and is not intended to restrict the scope of protection of the invention. As used at this point in the specification, including the appended claims, singular word forms, such as, for example, "a", "an", "the" or "the" include the plural equivalent, always and when the context does not specifically indicate otherwise. For example, the reference to "an antigen" includes a single antigen or a plurality of antigens, which in turn may be identical or different or the reference to "one method" includes equivalent steps and methods which are known to the person expert in the field.
The invention is explained in more detail below with reference to the non-limiting examples of specific modalities. The examples should be interpreted, in particular, as not being restricted to the combinations of features that are specifically illustrated, but instead the illustrative characteristics in turn can be freely combined as long as the object of the invention is achieved.
Example 1: Production of monodisperse silicon dioxide particles The production of the monodisperse silicon dioxide particles was carried out - as described in EP 0 216 278 Bl - by means of the hydrolysis of tetraalkoxysilanes in an aqueous-alcoholic-ammoniacal medium, where a The primary particle sol and the Si02 particles obtained are subsequently brought to the desired particle size by means of the continuous input measurement of tetraalkoxysilane in a controlled manner corresponding to the degree of reaction. The production of 50 g of Si02 particles having a size of 25 nm requires, for example, 1.2 1 of EtOH as a solubilizer, 860 ml of deionized water, 167 ml of tetraethyl orthosilicate (TEOS) and 28.5 ml of ammonia solution. watery to 25%. The spherical particles of silicon dioxide were determined by means of dynamic light scattering measurements using a Zetasizer Nano ZSMR device (Malvern Instruments, Herrenberg, Germany). The PDI-Malvern (polydispersity index) that had values < 0.1 showed a monodisperse distribution. Figure 1 illustrates the particle size and morphology by means of a SEM photomicrograph.
Example 2: Preparation of OVA SIINFEKL peptide fragment having an N-terminal alkyne group The peptide was constructed on a rink amide resin by means of Fmoc chemistry. Protected amino acids with N-alpha-Fmoc having suitable side chain protecting groups were employed. The solvent used was N-methylpyrrolidone. First, the peptide chain was created in an automatic synthesizer (Applied Biosystems Model ABI 433 A). After termination of the sequence, the terminal Fmoc protecting group was excised. The polymer was manually coupled to the alkynecarboxylic acid in a syringe. It was carefully washed with DMF, followed by dichloromethane and methanol and the resin was dried in vacuo overnight. For cleavage and deprotection, 5 ml of a mixture of TFA / H20 / phenol / triisopropylsilane (37: 1: 1: 1) was added to the resin and the mixture was stirred at room temperature for 2 hours. The TFA solution was transferred into a centrifuge tube and precipitated by slow addition of diethyl ether at 4 ° C, centrifuged, washed twice by the addition of diethyl ether, dried and taken in 2 ml of H20 / acetonitrile (1: 1 v / v). Purification was carried out by means of RP-HPLC using a select B column for RP (150 x 10 mm) with a gradient of 0% B-100% B in 7.5 minutes (A = H20 and B = acetonitrile, both comprising 0.1% TFA), flow rate = 10 ml / minute. The homogeneity and identity of the purified product was confirmed by means of analytical HPLC and mass spectrometry. After purification by RP-HPLC, the peptide was lyophilized.
Example 3: Functionalization of silicon dioxide particles with 3-bromopropyltrimethoxysilane 1 g of the Si02 particles (25 nm) produced in Example 1 was suspended in an ethanol / water mixture (100 ml, 4: 1) and 0.3 ml of 25% aqueous ammonia solution was added. 0.25 ml of the 3-bromopropyltrimethoxysilane (ABCR, Karlsruhe, Germany), dissolved in 10 ml of ethanol, were slowly added dropwise later via a dropping funnel and the mixture was heated under reflux for about 20 hours. The reaction mixture was cooled to room temperature and the Si02 particles were washed 5 x with an ethanol / water mixture (4: 1). All washing steps were carried out by centrifugation for 10 minutes at 9000 xg and 20 ° C in a temperature controlled centrifuge in 50 ml reaction vessels and by resuspension of the particles using an ultrasound finger .
Example 4: Reaction of the functionalized particles of silicon dioxide with sodium azide The Si02 particles functionalized with 3-bromopropyltrimethoxysilane in the third example were again dispersed in 80 ml of dimethyl sulfoxide (DMSO), 1 g of sodium azide and 100 mg of tetrabutylammonium bromide were added and the mixture was stirred at 80 ° C for 40 hours. 200 ml of deionized water were subsequently added and the particles were isolated by means of an ultrafiltration method using a membrane with a retention capacity of 10 kDa (Millipore, Bedford, USA) and washed with 600 ml of deionized water.
Example 5: Binding of OVA SIINFEKL peptide fragment to functionalized SiQ2 particles The azide-Si02 particles produced in the fourth example were resuspended in 40 ml of acetonitrile, the OVA peptide fragment of Example 1 (SIINFEKL-alkyne), diisopropylethylamine (DIPEA) and copper iodide (I) were added and the mixture was added. it was stirred at room temperature for about 20 hours. 100 ml of deionized water were added to the suspension, the product was isolated by means of ultrafiltration via a 10 kDa membrane (Millipore, Bedford, USA) and washed with 200 ml of deionized water and 50 ml of aqueous solution of EDTA.
Example 6: Testing of silica nanoparticles for adjuvant activity in vivo using PBL (peripheral blood lymphocyte) phenotypes as reading The investigations were carried out in C57B1 / 6 mice. The animals were divided into 3 groups (2 mice per group) to which they were administered either PBS (phosphate buffered saline), LPS (lipopolysaccharide) or silica nanoparticles (25 nm). The PBS served as control and the LPS as a reference (TLR4 agonist) compared against the silica nanoparticles with respect to their adjuvant activity. In the experiment, unmodified silica nanoparticles having a size of 25 nm were investigated which were produced by the method described in Example 1, dialyzed and subsequently filtered under sterile conditions. The silica dispersion was subsequently investigated by the endotoxins in order to ensure that the reading of the experiment in animals would not be falsified by the endotoxin contamination of the nanoparticles. The concentration of endotoxins in the investigated nanoparticle dispersion was lower than the maximum level recommended by Ph.Eur. for parenteral, liquid formulations of 0.5 IU / ml. 100 μ? of each of the test solutions or a dispersion were administered s.c. (by the subcutaneous route) to the animals on the flank. The nanoparticle dispersion comprised 450 μg of silica nanoparticles in 100 μ? of PBS. For reference, 10 of LPS were administered. 75-100 μ? of peripheral blood / mouse were taken by retro-orbital bleeding by means of a heparinized capillary tube and collected at heparinized rates of Eppendorf. The blood samples were labeled with several detection cocktails. The percentage distribution of immunologically relevant PBL phenotypes was subsequently determined by means of FACS (fluorescence activated cell sorting). In. Figures 2a-2c, the percentage of proportion of the four subpopulations of PBL CD4 (Figure 2a), CD8 effector (Figure 2b), CDllb + and CDllc + (DC) (Figure 2c) with respect to the total population of PBL was plotted on a graph. The data confirm that, after administration of the silica nanoparticles, the number of immunologically important T cells and dendritic cells was increased compared to the vehicle control PBS, indicating an adjuvant effect of the silica nanoparticles.
Example 7; Tagged with 99mTc of silica nanoparticles in water The nanoparticle solution (25 nm, solid content 9.0 mg / ml) was filtered through a 0.22 μ? Filter unit to MILEX-GV before use. 50 μ? of silica nanoparticles were added to 99mTc (132 MBq in 40 μ?) and the solution was mixed. 2 μ? of a solution of SnCl2 (0.1% of SnCl2 dihydrate in 10 mM NC1) were then added and the solution mixed again. After approximately 2 minutes, 150 μ? of 0.5 M phosphate buffer pH 8 were added and the solution was transferred into a Millipore Microcon Ultracel YM-100 centrifuge filter device and centrifuged at 13,000 rpm for 3 minutes. The filter was washed twice with 200 μ? of phosphate buffer 0.5 M pH 8 each time. The total filtrate comprised 46.84 MBq of 99mTc. 69.8 BMq of 99mTc remained in the filter. The particles in the filter were suspended twice in 200 μ? of phosphate buffer 0.5 M pH 8 each time and recovered by means of the filter rotation and short centrifugation. A particle suspension labeled with 28 MBq of 99mTc was obtained. The suspension of particles obtained was subsequently used for the experiment in animals.
Example 8: Formation of in vivo images of silica nanoparticles labeled with 99mTc The particles were labeled as described in Example 7. Non-invasive imaging of sentinel lymph node migration was subsequently carried out. For this purpose, istar female rats weighing approximately 400-500 g were anesthetized by inhalation anesthesia using isoflurane. The animals were given a subcutaneous injection of a clear suspension of the radiolabelled particles comprising 10-20 MBq of 99mTc in one of the two hind legs. The animals, even under anesthesia, were subsequently investigated by means of scintigraphy. For this purpose, the accumulation in the lower body of the animals at various times was exhibited using a gamma scintillation camera. The kinetics of these photographs show a significant accumulation of the particles in the lymph nodes leading away from the hind paw (see Figure 3). These are sentinel lymph nodes. In addition, a transient accumulation in the kidneys and bladder is evident. In contrast, the accumulation in the lymph node is continuous for 24 hours. Migration rates of approximately 1% of the administered dose were observed. As a control experiment, migration analysis of Nano-Albumon ™ labeled with 99mTc, a commercially available colloidal preparation for sentinel lymph node imaging, was carried out. The accumulation of this substance was comparable to that of the silica particles. By means of an additional control experiment, in which free 99mTc was administered by means of a subcutaneous injection into one of the two hind legs of female Wistar rats, it was ensured that free 99mTc did not accumulate in the lymph node. In this way, it was shown that silica nanoparticles are capable of targeting the lymph nodes, whereby the activation of the immune system can take place.
The following examples relate to pharmaceutical preparations.
Example A: Flasks for injection A solution of 100 g of nanoparticles and 5 g of disodium acid phosphate in 3 1 of distilled water is adjusted to pH 6.8 using 2 N hydrochloric acid, filtered under sterile conditions, transferred into vials for injection, lyophilized under conditions sterile and sealed under sterile conditions. Each vial for injection contains 5 mg of nanoparticles.
Example B: Suppositories A mixture of 20 g of nanoparticles with 100 g of soy lecithin and 1400 g of cocoa butter is melted. Pour into molds and let cool. Each suppository contains 20 mg of nanoparticles.
Example C: Solution A solution is prepared from 1 g of nanoparticles, 9.38 g of NaH2P04 * 2 H20, 28.48 g of Na2HP04 * 12 H2O and 0.1 g of benzalkonium chloride in 940 ml of bidistilled water. The H is adjusted to 6.8 and the solution is made up to 1 1 and sterilized by irradiation. This solution can be used in the form of eye drops.
Example D: Ointment 500 mg of nanoparticles are mixed with 99.5 g of Vaseline under aseptic conditions.
Example E: Tablets A mixture of 1 kg of nanoparticles, 4 kg of lactose, 1.2 kg of potato starch, 0.2 kg of talc and 0.1 kg of magnesium stearate is pressed to provide tablets in a conventional manner such that each tablet contains 10 mg of nanoparticles .
Example F: Dragees The tablets are pressed analogously to Example E and subsequently coated in a conventional manner with a coating of sucrose, potato starch, talc, tragacanth and dye.
Example G: Capsules 2 kg of nanoparticles are introduced into hard gelatin capsules in a conventional manner such that each capsule contains 20 mg of the nanoparticles.
Example H: Ampoules A solution of 1 kg of nanoparticles in 60 1 of bidistilled water is filtered under sterile conditions, transferred into ampoules, lyophilized under sterile conditions and sealed under sterile conditions, each vial contains 10 mg of nanoparticles.
Example I: Spraying for inhalation 14 g of nanoparticles are dissolved in 10 1 of isotonic NaCl solution and the solution is introduced into commercially available spray vessels with a pump mechanism. The solution can be sprayed into the mouth or nose. A spray shot (approximately 0.1 ml) corresponds to a dose of approximately 0.14 mg.
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:
1. Nanoparticles for parenteral use, characterized in that they comprise a matrix comprising more than 50%. of silicon dioxide, wherein the particles have a size of 5 to 50 nm and at least one cleavable connector as a surface functionality to which at least one antigen is covalently linked.
2. The nanoparticles according to claim 1, characterized in that the cleavable linker is a H-sensitive link, an enzymatic interface and / or an oxidising-cleavable linker, preferably a protease-sensitive peptide sequence or a hydrazone linker.
3. The nanoparticles according to one of the preceding claims, characterized in that they are monodisperse with a maximum standard deviation of 15%.
4. A process for the production of nanoparticles according to one of claims 1 to 3, characterized in that it has the following steps: (a) the hydrolytic polycondensation of tetraalkoxysilanes and / or organotrialkoxysilanes in a medium which comprises water, at least one solubilizer and at least one amine or ammonia, where first a primary particle sol and the resulting nanoparticles are produced. subsequently carry the desired particle size in a range of 5 to 50 nm such that additional nucleation is prevented by means of the continuous silane continuous input measurement in a controlled manner corresponding to the degree of reaction, (b) functionalization of the surface with a scissile connector as surface functionality, and (c) the covalent binding of an antigen to the functionality of the surface of the nanoparticles.
5. The process according to claim 4, characterized in that the non-porous nanoparticles are brought to the desired particle size in a range between 10 and 30 nm.
6. A dispersion, characterized in that it comprises the nanoparticles according to one of claims 1 to 3.
7. A pharmaceutical composition, characterized in that it comprises the nanoparticles according to one of claims 1 to 3 and / or the dispersion according to claim 6.
8. The use of nanoparticles according to one of claims 1 to 3 and / or of the dispersion according to claim 6 for the preparation of a vaccine for the targeting of antigens in antigen presenting cells, for the activation of the immune system and as an adjuvant.
9. The nanoparticles according to one of claims 1 to 3 and / or the dispersion according to claim 6, characterized in that they are for the targeting of antigens in antigen presenting cells, for activation of the immune system and as adjuvants.
10. The nanoparticles and / or dispersion according to claim 9, characterized in that they are for the prophylaxis or therapy of diseases selected from the group of infectious diseases, septic shock, tumors, cancer, autoimmune diseases, allergies and chronic or acute inflammation processes, preferably for the prophylaxis or therapy of tumors and / or cancer.
11. The dispersion according to claim 9 or 10, characterized in that it is for the targeting of the antigens in dendritic cells in lymph nodes, where the nanoparticles are monodisperse and not aggregated.
12. The nanoparticles, characterized in that they comprise a matrix comprising more than 50% silicon dioxide which has at least one surface functionality to which at least one antigen binds, where the particles have a size of 5 to 50 nm, for the targeting of antigens in cells that present antigens, for the activation of the immune system and as adjuvants.
13. A method for targeting and releasing antigens in antigen presenting cells, characterized in that it has the following steps: (a) the provision of nanoparticles according to one of claims 1 to 3, wherein the matrix comprises essentially pure silicon dioxide, (b) administration of the nanoparticles to cells that present antigens that are present in a culture of N cells, tissue, organs or a mammal, (c) the targeting of the antigens in antigen-presenting cells via the interstitial fluid by adjusting the targeting efficiency via the size of the nanoparticles, which are inversely proportional, (d) the uptake of the nanoparticles in the cells that present antigens and (e) the release of antigens from the matrix of the nanoparticles in the endosome.
14. The method according to claim 13, characterized in that, in step (a), the nanoparticles are produced by means of the process according to claim 4 and are provided in a dispersion.
15. A method of vaccination in the prophylaxis or therapy of diseases selected from the group of infectious diseases, septic shock, tumors, cancer, autoimmune diseases, allergies and chronic or acute inflammation processes, characterized in that an effective amount of the nanoparticles conforms to one of claims 1 to 3 and / or the dispersion according to claim 6 is administered to a mammal in need of this treatment.
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